Jiang, J.; Jones, C.; Reid, B.; Tsikritsis, D.; Mingard, K.; Ghai, P.; Kurttila, M.; Shaw, M. J.

High-resolution microscopy techniques are used across research and industry to analyse biological systems, from biomolecules to subcellular organelles, multicellular models and tissues. As multimodal imaging workflows and quantitative analysis of bioimaging data become increasingly widespread, there is a growing need for materials and methods to calibrate imaging systems and evaluate the fidelity of generated image data. Here, we present three-dimensional microscopy phantoms fabricated using two-photon photolithography from transparent resins that exhibit both broadband visible autofluorescence and Raman scattering across the fingerprint and C-H stretching regions. Suitable for analysis using optical profilometry, the phantoms were dimensionally calibrated with SI traceability using a metrological confocal microscope. Immersible in air and common aqueous imaging media, the phantoms are compatible with a wide variety of optical microscopy techniques, including one and two-photon excited fluorescence and coherent Raman scattering microscopy. We employed a forked wedge design to validate image deconvolution results and a stacked lattice phantom to recover image distortion matrices under realistic biological imaging conditions. We demonstrate the impact of correcting chromatic offsets and axial scaling errors for a representative application: analysis of a cell seeded scaffold using confocal laser scanning fluorescence microscopy. These phantoms provide a versatile platform for calibration, quality control and validation of multimodal imaging pipelines and improved quantitative optical microscopy.